1. Technical Field
The present invention relates to biochips and target DNA quantitative methods used for quantitative analysis of target DNAs.
2. Related Art
Methods of chemical analysis, chemical synthesis, and other procedures including various analyses in bio-related fields using a microfluidic chip that includes microchannels in a glass substrate or the like have gained attention. The microfluidic chip also has other names, such as a micro total analytical system (micro TAS) and a lab-on-a-chip. Because of advantages such as smaller amounts of samples and reagents, shorter reaction time, and less waste over common analyzing devices, the microfluidic chip is expected to have a wide range of applications, including medical diagnoses, on-site analyses of environment and food, and production of drugs and chemicals. Because the microfluidic chip requires small amounts of reagents, the amounts of expensive reagents can be reduced, and thus the cost of tests can be reduced. Requiring small amounts of samples and reagents means a shorter reaction time and improved test efficiency. Because samples such as blood are used in small amounts, the use of the microfluidic chip for medical diagnoses advantageously reduces the burden on patients.
The PCR (Polymerase Chain Reaction) method is well known as the method of amplifying nucleic acids, including DNA and RNA. In the PCR method, a mixture of target DNA and reagents is put into a tube, and a reaction is allowed in several minutes of repeated cycles at three different temperatures, for example, 55° C., 72° C., and 94° C., using a temperature control device called a thermal cycler. The enzyme (DNA polymerase) acts only on the target DNA and amplifies it about two-fold in each temperature cycle.
Real-time PCR, a technique that uses specific fluorescent probes to quantity DNA as it is amplified, has been put to practical applications. Because of high measurement sensitivity and reliability, real-time PCR has been widely used in research and clinical tests.
In real-time PCR, however, the amplification inhibitors present in a sample may lower the reliability of the results.
Simultaneous measurements of large numbers of reaction systems are usually difficult, because the amounts of DNA extracted from a sample are generally small, and the reagents used for the PCR are usually expensive. The amount of reaction mixture required for PCR is typically several ten microliters, and basically only a single gene can be assayed per reaction system.
WO/2005/059548 describes a method for assaying nucleic acids with the use of a fluorescent probe that hybridizes with both the target DNA and an internal standard DNA. Because this method coamplifies the target DNA and the internal standard DNA, the influence of amplification inhibitors is small, and an expensive, dedicated real-time PCR apparatus is not necessary. However, the method greatly suffers from poor quantification accuracy, particularly when there is a large concentration difference between the target DNA and the internal standard DNA, and the quantifiable concentration range of the target DNA is small.